RNA Decoys for DNA Binding Proteins

越来越多的人认识到，小的非编码rna可以参与基因调控。小rna能抑制dna结合蛋白吗？我们开发了一个人工例子，通过进行体外遗传选择实验，鉴定了一个小RNA适体，它在体外竞争性地抑制人类转录因子NF-kappaB与DNA的结合。酵母体内遗传选择优化导致RNA抑制NF-kappaB在活酵母细胞。我们已经解决了这种不寻常的RNA/NF-I<B复合物的x射线共晶结构。该结构证明了RNA结构可塑性如何允许DNA模仿，提出了一种针对不同NF-kappaB二聚体的工程RNA的新策略

There is growing appreciation that small, non-coding RNAs can participate in gene regulation. Can small RNAs inhibit DNA-binding proteins? We have developed an artificial example by performing in vitro genetic selection experiments identifying a small RNA aptamer that competitively inhibits human transcription factor NF-kappaB binding to DNA in vitro. Optimization by yeast in vivo genetic selections resulted in an RNA that inhibits NF-kappaB in living yeast cells. We have solved the X-ray co-crystal structure of this unusual RNA/NF-I<B complex. This structure demonstrates how RNA structural plasticity allows DNA mimicry, suggests a new strategy for engineering RNAs specific for different NF-kappaB dimers, and identifies a novel "open" NF-I<B conformation incompatible with DNA binding. NF-kappaB plays an essential role in regulating genes involved in inflammatory responses and protects tumor cells from apoptosis, thus limiting the effectiveness of chemotherapy and radiotherapy. Agents that reduce NF-kappaB activity tend to promote apoptosis and have shown promising anticancer activities. Using NF-kappaB as a model of therapeutic interest, we will optimize and test anti-NF-kappaB RNA aptamers in bacterial, yeast, and human cell culture experiments. We will also determine the structure of the free RNA aptamer to understand conformational changes that occur upon protein binding. Our unprecedented RNA/NF-kappaB crystal structure now allows virtual drug screens and in vitro and in vivo validation assays for novel NF-KappaB inhibitors. Such drugs would act by stabilizing our newly-discovered "open" NF-kappaB conformation that is incompatible with DNA binding. Four specific aims are proposed: Aim 1. Optimize and extend anti-NF-kappaB RNA aptamer function by genetic selections in bacteria and yeast. Aim 2. Analyze the structural basis for NF-kappaB binding by anti-NF-kappaB RNA aptamers. Aim 3. Analyze NF-kappaB inhibition by anti-NF-kappaB RNA aptamers in cultured mammalian cells. Aim 4. Identify small molecules that inhibit DNA binding by NF-kappaB.